Lipopolysaccharide (LPS) is a unique glycolipid found exclusively in the outer leaflet of the outer membrane of Gram-negative bacteria. Structurally1a,b, bacterial LPS molecule has three main regions: the O-antigen region, the core region and the Lipid-A region. The O-antigen region is a strain-specific polysaccharide moiety and determines the antigenic specificity of the organism. The core region is an oligosaccharide chain and may play a role in maintaining the integrity of the outer membrane. The Lipid-A region is conserved and functions as a hydrophobic anchor holding lipopolysaccharide in place.
LPS is known to trigger many pathophysiological events in mammals, either when it is injected or when is accumulated due to Gram-negative bacterial infection1b. Before the discovery of Lipid-A component of LPS the term “endotoxin” was generally used to describe the effects of the LPS. The endotoxin from Gram-negative bacteria is heat-stable, cell associated, pyrogenic and potentially lethal. In addition to its endotoxic activities, LPS also exhibits various biological activities, which include immuno adjuvant activity, B-lymphocyte mitogenesis, macrophage activation, interferon production, tumor regression, etc. While both the O-antigen and the core regions modulate the toxic activity of the LPS, it is generally believed that the hydrophobic Lipid-A moiety is responsible for these pathophysiological effects of the endotoxin2a, b.
Lipid-A consists of a β-(1,6)-linked D-glucosamine disaccharide phosphorylated at 1-O- and 4′-O-positions. Hydroxylated and non-hydroxylated fatty acids are linked to the hydroxyl and amino groups of the disaccharide to confer hydrophobicity to the Lipid-A. FIG. 1 shows two examples of natural Lipid-A structures, compound A2a,b isolated from E. coli, and compound B4a-d isolated from Salmonella strains. Takada and Kotani have conducted a thorough study of structural requirements of Lipid-A for endo-toxicity and other biological activities5a, thanks to the availability of synthetic Lipid-A analogs due to the efforts of various groups6-10. Ribi et al5b showed that the minimal structure required for toxicity was a bisphosphorylated β-(1,6)-linked di-glucosamine core to which long chain fatty acids are attached. It appears that an optimal number of lipid chains, in the form of either hydroxy acyl or acyloxyacyl groups, are required on the disaccharide backbone in order to exert strong endotoxic and related biological activities of Lipid-A6. For immunoadjuvant activity, however, the structural requirements of Lipid-A do not appear to be as rigid as those required for endotoxic activity and IFN-α/β or TNF-inducing properties5. Removal of all fatty acids, however, abrogates all biological activities normally attributed to Lipid-A.
In addition, removal of either phosphate group results in significant loss of toxicity without a corresponding loss of adjuvant activity. Bioassays on monophosphoryl Lipid-A showed that, while it was 1000 times less potent on a molar basis in eliciting toxic and pyrogenic responses, it was comparable to diphosphoryl Lipid-A (and endotoxin itself) in immunostimulating activities11a. It is known that the diphosphoryl Lipid-A from E. coli and Salmonella strains are highly toxic, but the monophosphoryl Lipid-A from E. coli has reduced toxicity while retaining the numerous biological activities that are normally associated with LPS11b, c, d.
The potent biological activities of Lipid-A have directed numerous research efforts toward developing useful applications. For example, the inhibition of Lipid-A biosynthesis is a new target activity for antibacterial drugs12, 13, and the drugs of future that function through this inhibitory mechanism will constitute a new class of antibiotics14, 15. The immunostimulating activity of Lipid-A has been investigated in order to develop new therapeutic anti-tumor agents16, 17 and immunoadjuvants by using modified Lipid-A structures and analogs. Furthermore, therapeutic agents of Lipid-A analogs have been investigated for treatment of sepsis18 based on their abilities to inhibit the interaction with macrophages, and as antagonists for the toxic activity of Lipid-A. Recently, Eisai19 developed a potent synthetic Lipid-A antagonist for the treatment of sepsis.
There is a need for effective treatment for Lipid-A/LPS associated disorders, and for a potent adjuvant without the associated toxicity. The high toxicity of unmodified Lipid-A from natural source prevents its general use as a pharmaceutical. A major drawback with the naturally derived Lipid-A is in accessing sufficient material with pharmaceutically acceptable purity, reproducible activity and stability. Naturally derived Lipid-A is a mixture of several components of cell wall including those of Lipid-A with varying number of lipid chains. Such heterogeneity in natural Lipid-A product is attributed to two sources: (1) biosynthetic variability in the assembly of the Lipid-A moiety and (2) loss of fatty acids from Lipid-A backbone during processing and purification. Consequently, it is difficult to control the manufacturing process in terms of reproducibility of composition of the mixture, which has significant bearing in biological activity and toxicity. For example, a reduction in adjuvant activity leads to reduction in immune response to an antigen that is formulated with Lipid-A as an adjuvant. The loss of a significant number of lipid chains during the processing of natural Lipid-A could result in the loss of adjuvant and other biological activities. Thus, it appears that lipid chains of Lipid-A molecules play a significant role in adjuvanticity such as internalization of antigens into macrophages and other antigen presenting cells (APC), leading to powerful immune responses.
While it is recognized that Lipid-A analogs are structurally complex, chemical synthesis is perhaps the best alternative to overcome the difficulties associated with accessing Lipid-A from natural sources. Natural combination of lipids refers to the lipid diversity that exists in nature. There is no lipid diversity in the synthetic lipids of present invention as they carry a uniform of contingent of lipids, which are of similar carbon length. The present invention relates to the design and synthesis of some new mono-phosphorylated Lipid-A analogs, each carrying a combination of unnatural lipids, such as (I) and (II) (FIG. 3) or significantly, an unnatural combination of lipids (FIG. 4). The following features distinguish the synthetic Lipid-A structures disclosed in this invention from those obtained from natural sources and/or reported in the prior art in the field.                1) Mono-phosphorylated: A chemically unmodified Lipid-A structure from nature carries two phosphate groups at 1- and 4′-position, while the synthetic Lipid-A analogs in the present invention carry one phosphate group at 4′-position.        2) A combination of unnatural lipids: Molecules, such as compounds 33 and 102 (FIG. 3), contain at least one novel and unnatural lipid (I or II).        3) An unnatural combination of lipids: This refers to those Lipid-A analogs that carry lipids of uniform chain length, a combination that is not found in nature and their synthesis is not known in the prior art. Compounds 54 and 86 (FIG. 4) fall into this category. Compound 70 (FIG. 19) is similar, but it also contains an n-propyl group at 3-O-position and is an example of Lipid-A analog that incorporates a short unnatural alkyl group with an unnatural ether linkage.        
All synthetic Lipid-A structures disclosed in this invention are expected to be mimics of naturally occurring E. coli derived and/or Salmonella derived Lipid-A structures (FIG. 1).